Polyphenols are widely distributed in plants, fruits, and vegetables and have received considerable attention because of their physiological functions in human and animal health, including antioxidant, antimutagenic and cancer prevention activities (Salvia et al., J. Agric. Food Chem. 39: 1549-1552, 1991; Bomser et al., Cancer Lett., 135: 151-157, 1999; Zhao et al., Carcinogenesis, 20: 1737-1745, 1999). Epidemiological studies have suggested that flavonoids, among the polyphenols, may reduce the risk of heart disease (Hertog et al., Lancet: 342: 1007-1011, 1993). Additionally, dietary flavan-3-ols and/or proanthocyanidins have been shown to reduce the incidence of atherosclerosis and coronary heart disease in experimental animals (Tijburg et al., Atherosclorosis, 135: 37-47, 1997; Yamakoshi et al., Atherosclerosis, 142: 139-149, 1999). One of the mechanisms responsible for these effects involves their inhibition of oxidation of low density lipoprotein (LDL) (Steinberg, Circulation, 85: 2337-2344, 1992).
The seeds of the cacao plant (Theobroma cacao L., Sterculiaceae) are known to be rich in polyphenols (Porter et al., Phytochemistry, 30: 1657-1663, 1991). Some of the antioxidant components of cacao liquor prepared from fermented and roasted cacao beans, which is a major ingredient of cocoa and chocolate products, have been characterized as flavan-3-ols and procyanidin oligomers (Sanbongi et al., J. Agric. Food Chem., 46: 454-457, 1998; Adamson et al., J. Agric. Food Chem., 47: 4184-4188, 1999).
Other species of Theobroma and other genera such as Herrania are also known sources of cocoa procyanidins. Twenty different species of Theobroma have been described but usually only 12 are accepted. Of these, nine are native to Amazonia, hence the center of genetic distribution appears to be the western half of the region (Giacometti, 1994, In “Neglected Crops: 1492 from a different perspective (J. E. Hernando Bermejo and J. Leon, Eds.) Plant Production and Protection Series No. 26, FAO, Rome, Italy, p205-209).
The genus Theobroma is typically neotropical and is distributed in the tropical rain forest in the Western Hemisphere between lat. 18° N and 15° S. The region with the most species is between Costa Rica and northeastern Colombia. Five sections and 20 species are recognized. Theobroma grandiflorum belongs to the section Glossopetalum, made up of 11 species; Theobroma cacao is the only species of the Theobroma section.
Four species of Theobroma have been described as producers of edible flesh: Theobroma grandiflorum, Theobroma canumanense Pires & Froes, Theobroma subincanum Martius, (Cupui in Brazil and Cacau de monte in Colombia) and Theobroma tricolor Humb. & Bonpl., which is a small tree distributed from western Amazonia to southern Mexico. Chocolate is also made from the seeds of these species (Giacometti, 1994, In “Neglected Crops: 1492 from a different perspective (J. E. Hernando Bermejo and J. Leon, Eds.) Plant Production and Protection Series No. 26, FAO, Rome, Italy, p205-209). It has been shown that beans of several species of Theobroma and Herrania produce similar procyanidins and that these compounds can be extracted from the beans (Romanczyk et al., WO 97/36497).
The polyphenols in cocoa beans are stored in the pigment cells of the cotyledons. Depending on the amount of anthocyanins in those pigmented cells, also called polyphenol-storage cells, they are white to deep purple. Three groups of polyphenols can be distinguished in these cells: catechins or flavan-3-ols (˜37%), anthocyanins (˜4%), and proanthocyanidins (˜58%). The main catechin is (−)-epicatechin which constitutes up to 35% of total polyphenol content. Procyanidins (commonly referred to as proanthocyanidins) are mainly flavan-3,4-diols, that are 4→8 or 4→6 bound to condensed dimmers, trimers, or oligomers with epicatechin as the main extension sub-unit (Romanczyk et al., WO 97/36497).
The total amount of soluble polyphenols in the dried fat-free mass of fresh cocoa beans is 15 to 20% (equaling ˜6% in air dried cocoa beans, containing 54% fat and 6% moisture), and in fermented beans ˜5%. Thus, one of the major drawbacks of using cocoa beans as a source of polyphenols is that most of the polyphenols are lost during processing of the beans. Other steps such as roasting and defatting also lead to losses. Thus, cocoa powder has less than 10% of the total polyphenols found in fresh beans. Another problem for using cocoa beans is the limited growth range for the plant, Theobroma cacao. It grows only in warm, moist climates in areas about 20° latitude north and south of the equator. This makes it difficult to preserve the polyphenol content of the beans during storage and transportation to areas where they can be processed and polyphenols extracted.
Plant cell culture is an attractive alternative to overcome these problems. Plant cell cultures have recently been used for the isolation of flavonoids. In the case of procyanidins, several groups were able to isolate particular compounds from cultures. For example, 4→8 linked (−)-epicatechin-(+)-catechin and gallic acid have been isolated from a Rosa culture (Muhitch & Fletcher, Plant Physiol., 75:592-595, 1984). Suspension cultures and calluses of Cryptomeria japonica were found to produce as much as 26% of dry weight as procyanidins (Teramoto & Ishikura, Bot. Mag. Tokyo 98: 171-179, 1985; Ishikura & Teramoto, Agric. Biol. Chem. 47: 421-423, 1983), and Pseudotsuga mensiesii suspension cultures produced as much as 40% of their dry weight as procyanidins (Stafford & Cheng, Phytochemistry 19: 131-135, 1980). Reports have also shown the production of procyanidins in cell suspension cultures of Vitis vinifera (Decendit & Merillon, Plant Cell Rep. 15: 762-765, 1996; Waffo-Teguo et al., Phytochem. 42:1591-1593, 1996).
Tissue culture research in Theobroma cacao has focused on somatic embryogenesis, which has been developed in several laboratories for the purpose of clonal propagation of the plant. The first report of Theobroma cacao somatic embryogenesis was by Esan in 1977 (Proc. 5th Int. Cacao Res. Conf. 1975. Ibadan: Cacao Res. Inst. Nigeria, 1977: 116-125, 1977), who described a method using immature zygotic embryo tissue explants. Similar methods were later reported by others (Pence et al., J. Am. Soc. Hort. Sci. 104: 145-148, 1979; Villalobos & Aguilar, Abstr. VII Int. Congr. Plant Tissue and Cell Cult., Amsterdam, Int. Assoc. for Plant Tissue Culture, pp 140, 1990). Later studies were focused on development of tissue culture methods from somatic tissues including leaves (Litz, In Dimick, P.S., Ed., Cacao biotechnology symposium. The Pennsylvania State University Press, University Park, Pa., pp 111-120, 1986), nucellus (Chatelet et al., C.R. Acad. Sci., Paris 315: 55-62, 1992; Figueira & Janick, Acta Hort. 336: 231-238, 1993; Sondhal et al., Acta Hort. 336: 245-248, 1993), and floral explants including petals and staminodes (Lopez-Baez et al., C.R. Acad. Sci., Paris 316: 579-584, 1993; Alemanno et al., Plant Cell Tiss. Organ Cult. 46: 187-194, 1996; Alemanno & Michaux-Ferriere, In Vitro Cell Dev. Biol. Plant 33: 163-172, 1997). These early methods, though successful, were not applicable to all genotypes and the frequency of regenerated plants was low. More efficient methods capable of propagating a wide range of genotypes were also developed (Li et al., In Vitro Cell Dev. Biol. Plant 34: 293-299, 1998; Maximova et al., In Vitro Cell Dev. Biol. Plant 38: 252-259, 2002). However, all the described methods, while using tissue culture methods to produce somatic embryos, did not teach methods to raise suspension cells.
There is limited amount of published work on developing cell cultures of Theobroma cacao. Most of this work was in the 1970's and 1980's (Hall & Collin, Annals of Bot. 39: 555, 1975; Jalal & Collin, Phytochem. 16: 1377-1380, 1977; Jalal & Collin, New Phytol. 83: 343-349, 1979; Tsai et al., J. Food Sci. 47: 768-773, 1982; Wen et al., J. Am. Oil Chemist's Soc. 16: 1720-1724, 1984). Of these, only a few have studied flavonoids in cell cultures of Theobroma cacao. For instance, Jalal and Collin (1979) reported that the flavonoid compositions of the callus and cell suspensions were similar and much less varied than that of the original intact cotyledon. Both tissue cultures contained (−)-epicatechin, leucocyanidins, caffeic and coumaric acids. The methylated purines, theobromine and caffeine, could not be detected in the tissue cultures. However, Gurney et al. (J. Expt. Bot. 43: 769-775, 1992) reported that callus and suspension cultures of Theobroma cacao produced caffeine, theobromine and theophylline at concentrations about 10% of those found in vivo.
None of the prior art has reported the formation of oligomeric procyanidins, which in recent years have been shown to have the greatest bioefficacy. Jalal and Collin (New Phytol. 83: 343-349, 1979) reported the detection of leucocyanidins in cell cultures of Theobroma cacao. However, based on the methods used to detect the compounds it was not possible to characterize the nature or size of the leucocyanidins. Further, tissue culture methods have not been described for other species of Theobroma or Herrania. 